Cell-Specific α-Tubulin Isotype Regulates Ciliary Microtubule Ultrastructure, Intraflagellar Transport, and Extracellular Vesicle Biology

Abstract

Cilia are found on most non-dividing cells in the human body and, when faulty, cause a wide range of pathologies called ciliopathies. Ciliary specialization in form and function is observed throughout the animal kingdom, yet mechanisms generating ciliary diversity are poorly understood. The "tubulin code"-a combination of tubulin isotypes and tubulin post-translational modifications-can generate microtubule diversity. Using C. elegans, we show that α-tubulin isotype TBA-6 sculpts 18 A- and B-tubule singlets from nine ciliary A-B doublet microtubules in cephalic male (CEM) neurons. In CEM cilia, tba-6 regulates velocities and cargoes of intraflagellar transport (IFT) kinesin-2 motors kinesin-II and OSM-3/KIF17 without affecting kinesin-3 KLP-6 motility. In addition to their unique ultrastructure and accessory kinesin-3 motor, CEM cilia are specialized to produce extracellular vesicles. tba-6 also influences several aspects of extracellular vesicle biology, including cargo sorting, release, and bioactivity. We conclude that this cell-specific α-tubulin isotype dictates the hallmarks of CEM cilia specialization. These findings provide insight into mechanisms generating ciliary diversity and lay a foundation for further understanding the tubulin code.

Keywords: C. elegans; cilia; extracellular vesicles; glutamylation; intraflagellar transport; kinesin-3; microtubule; polycystin; post-translational modifications; tubulin.

Figure 1. Specialized ultrastructure of adult CEM cilia reveals a novel axonemal microtubule arrangement

A Left: Adult C. elegans male DIC image with region of interest boxed. Middle: Each of four quadrant cephalic sensilla contains the endings of CEM and CEP neurons and the socket and sheath support cells. Right: The cephalic sensillum contains cilia of the CEM and CEP neurons surrounded by socket (blue) and sheath (pink) cells. The CEM cilium (green) curves out and the tip protrudes to the environment via a cuticular pore (orange); while CEP cilium (gray) curves in and embeds in the cuticle. The cephalic lumen formed by the sheath and socket cells contains CEM-derived EVs [17]. B The CEM axoneme has five ultrastructurally distinct regions. Right: Serial electron tomogram model shows the relative positions of the five distinguishable regions. Sections labeled C-F correspond to subsequent panels. Left: TEM inserts show the representative microtubule organization. Scale bar, 250nm. dMT, doublet microtubule; sMT, singlet microtubule. Cartoon inserts show a stereotypical microtubule arrangement within each segment and do not directly reflect adjacent TEM sections. C Tomogram model of A- and B- tubules of a microtubule doublet splitting into an A-tubule and B-tubule singlet. Left: Longitudinal section taken from a serial electron tomogram depicting a representative microtubule doublet splitting into A- and B- tubule singlets at the proximal doublet region. Middle: segmented tomogram model of the left panel depicting microtubules in orange, and ciliary membrane is green. Teal rings depict the boundary of 500–600nm long region where most microtubule doublets split into two singlets. Numbers correspond to the equivalent regions in the tomogram and model. Right: tomogram cross-sections of a representative doublet splitting into two singlets. Refer to Figure S1 for images of all nine microtubules. D Some microtubules form bridges with the ciliary membrane. Left: flat-plane image taken from electron tomogram illustrating a microtubule singlet making three bridges with ciliary membrane. Right: density-based isosurface 3D model of this location. Middle: overlay. Scale bar, 50nm. Refer to Movie S2 for annotated 3D tomogram. In the 75nm cilia section examined, 5 out of 9 A-tubules, 6 out of 8 B-tubules, and 0 out of 2 inner singlets were connected the ciliary membrane at least at one point. Of those membrane-associated tubules, the number of connections was: 4.4±6.3 for an A-tubule and 11.16±3.9 for a B-tubule (mean±SD). The difference between A-tubule and B-tubule connections did not reach statistical significance E Tomogram model of the sub-distal region depicting A- and B- tubule singlet fusion. Left: segmented electron tomogram model depicting fusion of A-tubule and B-tubule singlets (pink and teal false-colored), and the A-tubule singlet continuing to the distal segment (incomplete). Ciliary membrane is green. Right: false-colored longitudinal section from the electron tomogram used to generate the model on the left. F B-tubules form transient C-shaped structures during the singlet-to-doublet transition in the sub-distal segment (refer to Figure 1B). Left panel: successive images from a tomogram. Scale bar, 50nm. Right panel: isosurface 3D models corresponding to images on the left. Middle panel: overlay. See also Figure S1, Movies S1 and S2.

Figure 2. tba-6 is required for CEM cilia curvature, microtubule architecture, and tubulin composition

A, B, B′ Serial TEM reconstructions of male cephalic sensilla of WT and tba-6 males. Pie charts (bottom) indicate the penetrance of the cilia curvature phenotype as assayed by TBB-4::tagRFP (WT N=83, tba-6 N=102 males). A In WT, CEM (yellow) and CEP cilia (gray) share a lumen formed by the sheath (inner border in pink) and socket (teal) (refer to Figure 1A). The distal tip of the CEM cilium protrudes to the environment through a cuticular pore (brown) (also, Figure S1C). The CEP cilium curves in and embeds in the cuticle. EVs (green) are located in the proximal region of the cephalic lumen. B 59% of tba-6 cilia curve-out but do not protrude through the cuticular pore. EVs were observed inside the cuticular pore (for TEM cross sections of tba-6, see Figure 4C). B′ 41% of tba-6 cilia curve-in, follow the path of the CEP cilium, and embed in the cuticle. EVs were found in the lumen surrounding CEM and CEP cilia. C In tba-6, microtubule doublets terminate and do not form A- and B- tubule singlets. Right: Serial TEM images of tba-6 cilia with A-tubules continuing as singlets and B-tubules terminating (black arrow). Left: Cross-sections of WT cilia from a serial tomogram showing a representative A- and B-tubule of a microtubule doublet splitting to form two singlets (white arrow) (refer to Figure 1F and Figure S1A, B). Scale bar, 50nm. D Cartoon depicting microtubule architectures and cilia shapes. E Cross sections of sub-distal CEM cilia in WT and tba-6 animals. Scale bar, 50nm. Right: Microtubule singlet quantification: WT= 20±2, tba-6 curved-out = 14±2 tubules, mean±SD, p-value=0.0269 by Mann Whitney, N=3 animals, and n=12 cilia for each genotype. F, F′, G, G′ β-tubulin TBB-4::GFP relative ciliary levels and ciliary localization. In WT, TBB-4::GFP localizes to CEM cilia in a stereotypical pattern; lines indicate two of four CEM cilia, quantified in G, n= 25 cilia. Ciliary TBB-4::GFP levels were normalized to levels in the CEM dendrite, mean fold intensity ± SD. This pattern is altered in tba-6 mutants, quantified in G′, n=20 cilia. In tba-6, TBB-4::GFP ciliary levels are significantly reduced and TBB-4::GFP accumulates at ciliary bases. F′, N=86 and 102 animals for WT and tba-6 respectively, p-value < 0.0001 by Mann-Whitney U test, Scale bar,10μm. See also Table S1, and Figure S2.

Figure 3. Localization and velocity distributions of IFT motors and polypeptides in CEM cilia of wild type and tba-6 mutant animals

A Widefield fluorescence images of GFP tagged IFT polypeptides and motors. Lines in each image indicate two of the four CEM cilia. OSM-3::GFP and KLP-6 GFP are driven by the klp-6 promoter and expressed in both CEM (arrows) and IL2 cilia. All other reporters are driven by the pkd-2 promoter and expressed in CEM cilia. Phenotypes are summarized in Table S1. Scale bar is 10μm and applicable to all panels. B Comparison of fluorescence levels in cilia of WT and tba-6 males normalized to cell body. Mean ± SD. ‘*’, ‘**’, ‘***’ indicate p-values of <0.01, <0.001 and, <0.0001 respectively by Mann-Whitney U test. C Average CEM ciliary localization pattern of KAP-1::GFP and OSM-3::GFP in WT and tba-6 mutant backgrounds. ‘◆’ indicates feature used to align intensity profiles; center-line depicts the mean intensity and the shaded area represent the standard deviation. D Velocity distributions of GFP-tagged IFT motors and polypeptides in CEM cilia of WT and tba-6 males. Distribution averages, standard deviations, and statistical analyses are summarized in Table 1. See also Table S1.

Figure 4. tba-6 regulates EV location, abundance, and size in the cephalic sensilla

A, B PKD-2::GFP and CIL-7::GFP abnormally accumulate in tba-6 mutant animals. Quantified on right. See Table S1 for statistical analysis. Lines indicate cilia. Scale bar, 10μm. C Simplified serial TEM reconstructions depicting relative positions of lumenal EVs. Genotypes are indicated at the top and respective reconstructions and TEM images are arranged vertically. Scale bars in TEM images are 500nm. C-top row The CEM cilium is red and distal dendrite is yellow. For simplicity, the cuticular pore is outlined using a dotted line and the CEP cilium is not shown. Dashed lines indicated by ‘Cu’(cuticular pore) or ‘P’ (proximal lumen) denote the relative position of the TEM cross sections. C-middle row TEM cross section at the cuticular pore. The WT distal CEM cilium is present and contains microtubule singlets. The tba-6 curved out CEM cilium does not reach the cuticular pore. Instead, EVs are seen in the empty pore. tba-6 curved in cilia are deflected from the pore and embedded in the cuticle lumen next to the CEP cilium. EVs are seen in the cephalic lumen and cuticle pore. C-bottom row TEM cross-sections from proximal sheath cell level. In WT, few EVs are observed in the cephalic lumen. In tba-6, EVs abnormally accumulate in the cephalic lumen. D and E Comparison of average number of EVs and average EV diameter in the distal and proximal cephalic lumen. In WT, EVs were more abundant in the proximal lumen (distal=14±10 EVs, proximal=80±60 EVs, p-value=0.0087, ‘*’, by Mann-Whitney U test). When present, distally located EVs were significantly smaller than proximally located EVs (distal=36.67±0.63nm, proximal=105±29.14nm, mean±SD, n=8 cilia, p-value<0.0001 for size, ‘***’, by Mann-Whitney U test). tba-6 accumulated significantly more lumenal EVs than WT (distal=252±10 and proximal=753±77 EVs, mean±SD, p-value=0.007 for both with WT using Kruskal-Wallis with Dunn’s correction). tba-6 distally located EVs were larger than WT distally located EVs (diameters were 61.65±13.96nm and 54.86±9.76nm for curved out (n=6) and curved in (n= 2) sensilla, p-value<0.0001 compared with WT by Kruskal-Wallis with Dunn’s correction). tba-6 proximally located EVs were smaller than WT proximally located EVs (diameters were 84.57±35.21nm and 71.48±19.75nm, mean±SD, for curved-out and curved–in sensilla, p-values<0.0001 compared with wild type by Kruskal-Wallis with Dunn’s correction). G and H Average histograms of EV diameter within the distal and proximal cephalic lumen in WT and tba-6. In tba-6 curved-in cilia 50–70nm sized EVs accumulate in the lumen (WT distal: 8 50–60nm EVs, tba-6 curved-in distal: 59 50–60nm EVs; WT proximal: 13 50–60nm EVs, tba-6 curved-in proximal: 346 50–60nm EVs n=8 sensilla for WT and n=2 sensilla for tba-6 curved-in, p-value<0.0001 by two-way ANOVA). I Occlusion of the CEM cilia tip correlates with luminal EV accumulation. In WT, CEM cilia tips are environmentally exposed through the cuticular pore (see Figure S1C) and contain 47±53 lumenal EVs, n=7 sensilla. Tips of the tba-6 curved-out cilia do not reach pore (hence “partial” label), accumulate 116±170 EVs (n=6 sensilla). Tips of tba-6 curved-in cilia are not environmentally exposed and accumulate on 502±272 lumenal EVs (n=4 sensilla). R² indicates ‘goodness of fit’ of the cubic polynomial line. Number of EVs reflect mean ± SD and n is number of cephalic sensilla analyzed via quantitative serial EM. See also Table S1

Figure 5. tba-6 is required for ciliary EV cargo composition, release, and bioactivity

A WT and tba-6 males release PKD-2::GFP containing EVs. B Cartoon depicts the single-focal-plane fluorescence image acquired at the surface of the cover slip showing PKD-2::GFP-containing EVs in WT and tba-6 worms. In A, arrowheads point to EVs; yellow brackets mark PKD-2::GFP EV streaks. Abundance of EV streaks is quantified below. Environmental release of PKD-2::GFP-containing EVs is significantly reduced in tba-6 mutants (WT: ‘0’ = 0, ‘≤10’= 0.04±21, ‘≤50,’= 0.34±0.48, ‘>50’=0.60±0.49, fraction ± SD, N=88 worms; tba-6: ‘0’ = 0.14±35, ‘≤10’= 0.30±0.46, ‘≤50’= 0.52±0.50, ‘>50’=0.05±0.21, fraction ± SD, N=86 worms). WT and tba-6 mutants are significantly different in ‘≤10’, ‘≤50’ and ‘>50’catogories, ‘*’ and ‘***’ indicate p-values of <0.01 and <0.0001, respectively, using the Kruskal-Wallis test with Dunn’s correction for multiple comparisons. Error bars indicate SEM. Fraction of animals with PKD-2::GFP-positive EV streaks was significantly reduced in tba-6 background (WT = 0.37±0.49, tba-6 = 0.10±0.34) ‘***’ indicates p-value <0.0001 using Mann-Whitney test. Fraction ± SD, N = 88 (WT) and 86 (tba-6) animals. Error bars indicate SEM. C In WT, KLP-6::GFP is excluded from EVs. In tba-6 mutants, KLP-6::GFP is ectopically shed and released into EVs. Arrowheads point to ectopic EVs (refer Figure 3). Quantified on right. Statistical details are summarized in Table S1. Scale bar is 10μm. D TBA-6 is required for bioactivity of EVs. Left: Young adult virgin WT males were individually placed on a freshly made EV-containing bacterial lawn and video recorded for 5 minutes. Inset Sample time series of tail-chasing behavior; in this case the male was tail-chasing from 117^th to 124^th second. Middle: WT males exhibit a basal level of tail chasing behavior (buffer control). WT-derived EVs increased the fraction of males exhibiting tail chasing (0.71±0.06 events/5 minutes vs buffer control 32±0.06 events/5 minutes, mean ± SEM, N=56 males and N=69 males for WT and buffer control, p-value<0.0001 with buffer control). In contrast, tail chasing events were similar with tba-6 derived EVs and buffer control (0.42±0.05 events/5 minutes, N= 69 tba-6 males, p-value>0.05 with buffer control and p-value<0.001 with WT EVs). Number within bars indicates number of animals. Letters indicate statistically distinct groups by Kruskal-Wallis test with Dunn’s correction for multiple comparisons. Error bars indicate SEM. Right: Number of tail chasing episodes per assay increases when WT males are exposed to WT EVs but not tba-6-derived EVs. See also Table S1

Figure 6. Model of a doublet microtubule, IFT motors and polypeptides in WT and tba-6 CEM cilia

In WT, microtubule doublets splay to form A- and B-tubule singlets. Heterotrimeric kinesin-II and IFT A- and B-polypeptides are co-transported at overlapping velocities. Homodimeric kinesin-2 OSM-3 travels at a higher velocity separate from IFT polypeptides, transporting unknown cargo. KLP-6 is not included in this model. In tba-6, microtubule doublets do not splay to form A- and B- tubule singlets. The doublet region is elongated and terminates abruptly. Only A-tubule singlets extend. Axonemal abnormalities accompany changes in IFT. IFT-A and –B polypeptides travel at distinct velocities that overlap with heterotrimeric kinesin-II and OSM-3, respectively. Red squares indicate possible TBA-6 sites of action. We propose a model where the separation of microtubule doublets into distinct A-tubule and B-tubule singlets acts as a physical substrate for the functional separation of these two conserved IFT kinesins. Alternatively, tubulin composition and post-translational modifications may also impact IFT-motor-cargo dynamics.